The neurofibromatosis type I pre-mRNA is a novel target of CELF protein-mediated splicing regulation

The CUG-BP and ETR-3 like factors (CELF) are a family of six highly conserved RNA-binding proteins that preferentially bind to UG-rich sequences. One of the key functions of these proteins is to mediate alternative splicing in a number of tissues, including brain, heart and muscle. To fully understand the function of CELF proteins, it is important to identify downstream targets of CELF proteins. In this communication, we report that neurofibromatosis type I (NF1) exon 23a is a novel target of CELF protein-mediated splicing regulation in neuron-like cells. NF1 regulates Ras signaling, and the isoform that excludes exon 23a shows 10 times greater ability to down-regulate Ras signaling than the isoform that includes exon 23a. Five of the six CELF proteins strongly suppress the inclusion of NF1 exon 23a. Over-expression or siRNA knockdown of these proteins in cell transfection experiments altered the levels of NF1 exon 23a inclusion. In vitro binding and splicing analyses demonstrate that CELF proteins block splicing through interfering with binding of U2AF⁶⁵. These studies, combined with our previous investigations demonstrating a role for Hu proteins and TIA-1/TIAR in controlling NF1 exon 23a inclusion, highlight the complex nature of regulation of this important alternative splicing event.     

Muscleblind-like 1 activates insulin receptor exon 11 inclusion by enhancing U2AF65 binding and splicing of the upstream intron

Alternative splicing regulates developmentally and tissue-specific gene expression programs, disruption of which have been implicated in numerous diseases. Muscleblind-like 1 (MBNL1) regulates splicing transitions, which are disrupted on loss of MBNL1 function in myotonic dystrophy type 1 (DM1). One such event is MBNL1-mediated activation of insulin receptor exon 11 inclusion, which requires an intronic enhancer element downstream of exon 11. The mechanism of MBNL1-mediated activation of exon inclusion is unknown. We developed an in vitro splicing assay, which robustly recapitulates MBNL1-mediated splicing activation of insulin receptor exon 11 and found that MBNL1 activates removal of the intron upstream of exon 11 upon binding its functional response element in the downstream intron. MBNL1 enhances early spliceosome assembly as evidenced by enhanced complex A formation and binding of U2 small nuclear ribonucleoprotein auxiliary factor 65 kDa subunit (U2AF65) on the upstream intron. We demonstrated that neither the 5′ splice site nor exon 11 sequences are required for MBNL1-activated U2AF65 binding. Interestingly, the 5′ splice site is required for MBNL1-mediated activation of upstream intron removal, although MBNL1 has no effect on U1 snRNA recruitment. These results suggest that MBNL1 directly activates binding of U2AF65 to enhance upstream intron removal to ultimately activate alternative exon inclusion.     

Alternative splicing of the neurofibromatosis type I pre-mRNA

NF1 (neurofibromatosis type?I) is a common genetic disease that affects one in 3500 individuals. The disease is completely penetrant but shows variable phenotypic expression in patients. NF1 is a large gene, and its pre-mRNA undergoes alternative splicing. The NF1 protein, neurofibromin, is involved in diverse signalling cascades. One of the best characterized functions of NF1 is its function as a Ras-GAP (GTPase-activating protein). NF1 exon 23a is an alternative exon that lies within the GAP-related domain of neurofibromin. This exon is predominantly included in most tissues, and it is skipped in CNS (central nervous system) neurons. The isoform in which exon 23a is skipped has 10?times higher Ras-GAP activity than the isoform in which exon 23a is included. Exon 23a inclusion is tightly regulated by at least three different families of RNA-binding proteins: CELF {CUG-BP (cytosine-uridine-guanine-binding protein) and ETR-3 [ELAV (embryonic lethal abnormal vision)-type RNA-binding protein]-like factor}, Hu and TIA-1 (T-cell intracellular antigen 1)/TIAR (T-cell intracellular antigen 1-related protein). The CELF and Hu proteins promote exon 23a skipping, while the TIA-1/TIAR proteins promote its inclusion. The widespread clinical variability that is observed among NF1 patients cannot be explained by NF1 mutations alone and it is believed that modifier genes may have a role in the variability. We suggest that the regulation of alternative splicing may act as a modifier to contribute to the variable expression in NF1 patients.     

U1 snRNP-dependent function of TIAR in the regulation of alternative RNA processing of the human calcitonin/CGRP pre-mRNA

Alternative RNA processing of human calcitonin/CGRP pre-mRNA is regulated by an intronic enhancer element. Previous studies have demonstrated that multiple sequence motifs within the enhancer and a number of trans-acting factors play critical roles in the regulation. Here, we report the identification of TIAR as a novel player in the regulation of human calcitonin/CGRP alternative RNA processing. TIAR binds to the U tract sequence motif downstream of a pseudo 5' splice site within the previously characterized intron enhancer element. Binding of TIAR promotes inclusion of the alternative 3'-terminal exon located more than 200 nucleotides upstream from the U tract. In cells that preferentially include this exon, overexpression of a mutant TIAR that lacks the RNA binding domains suppressed inclusion of this exon. In this report, we also demonstrate an unusual novel interaction between U6 snRNA and the pseudo 5' splice site, which was shown previously to bind U1 snRNA. Interestingly, TIAR binding to the U tract sequence depends on the interaction of not only U1 but also U6 snRNA with the pseudo 5' splice site. Conversely, TIAR binding promotes U6 snRNA binding to its target. The synergistic relationship between TIAR and U6 snRNA strongly suggests a novel role of U6 snRNP in regulated alternative RNA processing.     

Insights into the selective activation of alternatively used splice acceptors by the human immunodeficiency virus type-1 bidirectional splicing enhancer

The guanosine-adenosine-rich exonic splicing enhancer (GAR ESE) identified in exon 5 of the human immunodeficiency virus type-1 (HIV-1) pre-mRNA activates either an enhancer-dependent 5′ splice site (ss) or 3′ ss in 1-intron reporter constructs in the presence of the SR proteins SF2/ASF2 and SRp40. Characterizing the mode of action of the GAR ESE inside the internal HIV-1 exon 5 we found that this enhancer fulfils a dual splicing regulatory function (i) by synergistically mediating exon recognition through its individual SR protein-binding sites and (ii) by conferring 3′ ss selectivity within the 3′ ss cluster preceding exon 5. Both functions depend upon the GAR ESE, U1 snRNP binding at the downstream 5′ ss D4 and the E42 sequence located between these elements. Therefore, a network of cross-exon interactions appears to regulate splicing of the alternative exons 4a and 5. As the GAR ESE-mediated activation of the upstream 3′ ss cluster also is essential for the processing of intron-containing vpu/env-mRNAs during intermediate viral gene expression, the GAR enhancer substantially contributes to the regulation of viral replication.     

Exon‐independent recruitment of SRSF1 is mediated by U1 snRNP stem‐loop 3

SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5′ splice site (SS), and both factors affect 5′ SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single‐molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1‐independent process of binding to an ESE. Structural analysis and cross‐linking data show that SRSF1 contacts U1 snRNA stem‐loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5′SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals.     

Tra2-Mediated Recognition of HIV-1 5′ Splice Site D3 as a Key Factor in the Processing of vpr mRNA

Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3′ splice site (3′ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5′ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE_vpr) localized between exonic splicing silencer ESSV and 5′ss D3. The ESE_vpr sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE_vpr. Further analyses revealed that ESE_vpr supports the binding of U1 snRNA at 5′ss D3, allowing bridging interactions across the upstream exon with 3′ss A2. In line with this, an increase or decrease in the complementarity of 5′ss D3 to the 5′ end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3′ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5′ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3′ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.     

Functional studies on the ATM intronic splicing processing element

In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5′ and 3′ splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a ~40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5′–3′ order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing.     

Polypyrimidine Tract-Binding Protein Positively Regulates Inclusion of an Alternative 3′-Terminal Exon

Polypyrimidine tract-binding protein (PTB) is an abundant vertebrate hnRNP protein. PTB binding sites have been found within introns both upstream and downstream of alternative exons in a number of genes that are negatively controlled by the binding of PTB. We have previously reported that PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3′-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5′ splice site sequence to form a pseudoexon. Here we show that the binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB was antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminated enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation.     

Restriction of an intron size en route to endothermy

Ca²⁺-insensitive and -sensitive E1 subunits of the 2-oxoglutarate dehydrogenase complex (OGDHC) regulate tissue-specific NADH and ATP supply by mutually exclusive OGDH exons 4a and 4b. Here we show that their splicing is enforced by distant lariat branch points (dBPs) located near the 5′ splice site of the intervening intron. dBPs restrict the intron length and prevent transposon insertions, which can introduce or eliminate dBP competitors. The size restriction was imposed by a single dominant dBP in anamniotes that expanded into a conserved constellation of four dBP adenines in amniotes. The amniote clusters exhibit taxon-specific usage of individual dBPs, reflecting accessibility of their extended motifs within a stable RNA hairpin rather than U2 snRNA:dBP base-pairing. The dBP expansion took place in early terrestrial species and was followed by a uridine enrichment of large downstream polypyrimidine tracts in mammals. The dBP-protected megatracts permit reciprocal regulation of exon 4a and 4b by uridine-binding proteins, including TIA-1/TIAR and PUF60, which promote U1 and U2 snRNP recruitment to the 5′ splice site and BP, respectively, but do not significantly alter the relative dBP usage. We further show that codons for residues critically contributing to protein binding sites for Ca²⁺ and other divalent metals confer the exon inclusion order that mirrors the Irving-Williams affinity series, linking the evolution of auxiliary splicing motifs in exons to metallome constraints. Finally, we hypothesize that the dBP-driven selection for Ca²⁺-dependent ATP provision by E1 facilitated evolution of endothermy by optimizing the aerobic scope in target tissues.     

Extended base pair complementarity between U1 snRNA and the 5' splice site does not inhibit splicing in higher eukaryotes, but rather increases 5' splice site recognition

Spliceosome formation is initiated by the recognition of the 5′ splice site through formation of an RNA duplex between the 5′ splice site and U1 snRNA. We have previously shown that RNA duplex formation between U1 snRNA and the 5′ splice site can protect pre-mRNAs from degradation prior to splicing. This initial RNA duplex must be disrupted to expose the 5′ splice site sequence for base pairing with U6 snRNA and to form the active spliceosome. Here, we investigated whether hyperstabilization of the U1 snRNA/5′ splice site duplex interferes with splicing efficiency in human cell lines or nuclear extracts. Unlike observations in Saccharomyces cerevisiae, we demonstrate that an extended U1 snRNA/5′ splice site interaction does not decrease splicing efficiency, but rather increases 5′ splice site recognition and exon inclusion. However, low complementarity of the 5′ splice site to U1 snRNA significantly increases exon skipping and RNA degradation. Although the splicing mechanisms are conserved between human and S.cerevisiae, these results demonstrate that distinct differences exist in the activation of the spliceosome.     

The Pivotal Roles of TIA Proteins in 5′ Splice-Site Selection of Alu Exons and Across Evolution

More than 5% of alternatively spliced internal exons in the human genome are derived from Alu elements in a process termed exonization. Alus are comprised of two homologous arms separated by an internal polypyrimidine tract (PPT). In most exonizations, splice sites are selected from within the same arm. We hypothesized that the internal PPT may prevent selection of a splice site further downstream. Here, we demonstrate that this PPT enhanced the selection of an upstream 5′ splice site (5′ss), even in the presence of a stronger 5′ss downstream. Deletion of this PPT shifted selection to the stronger downstream 5′ss. This enhancing effect depended on the strength of the downstream 5′ss, on the efficiency of base-pairing to U1 snRNA, and on the length of the PPT. This effect of the PPT was mediated by the binding of TIA proteins and was dependent on the distance between the PPT and the upstream 5′ss. A wide-scale evolutionary analysis of introns across 22 eukaryotes revealed an enrichment in PPTs within ∼20 nt downstream of the 5′ss. For most metazoans, the strength of the 5′ss inversely correlated with the presence of a downstream PPT, indicative of the functional role of the PPT. Finally, we found that the proteins that mediate this effect, TIA and U1C, and in particular their functional domains, are highly conserved across evolution. Overall, these findings expand our understanding of the role of TIA1/TIAR proteins in enhancing recognition of exons, in general, and Alu exons, in particular.     

CA- and Purine-Rich Elements Form a Novel Bipartite Exon Enhancer Which Governs Inclusion of the Minute Virus of Mice NS2-Specific Exon in Both Singly and Doubly Spliced mRNAs

The alternatively spliced 290-nucleotide NS2-specific exon of the parvovirus minute virus of mice (MVM), which is flanked by a large intron upstream and a small intron downstream, constitutively appears both in the R1 mRNA as part of a large 5′-terminal exon (where it is translated in open reading frame 3 [ORF3]), and in the R2 mRNA as an internal exon (where it is translated in ORF2). We have identified a novel bipartite exon enhancer element, composed of CA-rich and purine-rich elements within the 5′ and 3′ regions of the exon, respectively, that is required to include NS2-specific exon sequences in mature spliced mRNA in vivo. These two compositionally different enhancer elements are somewhat redundant in function: either element alone can at least partially support exon inclusion. They are also interchangeable: either element can function at either position. Either a strong 3′ splice site upstream (i.e., the exon 5′ terminus) or a strong 5′ splice site downstream (i.e., the exon 3′ terminus) is sufficient to prevent skipping of the NS2-specific exon, and a functional upstream 3′ splice site is required for inclusion of the NS2-specific exon as an internal exon into the mature, doubly spliced R2 mRNA. The bipartite enhancer functionally strengthens these termini: the requirement for both the CA-rich and purine-rich elements can be overcome by improvements to the polypyrimidine tract of the upstream intron 3′ splice site, and the purine-rich element also supports exon inclusion mediated through the downstream 5′ splice sites. In summary, a suboptimal large-intron polypyrimidine tract, sequences within the downstream small intron, and a novel bipartite exonic enhancer operate together to yield the balanced levels of R1 and R2 observed in vivo. We suggest that the unusual bipartite exonic enhancer functions to mediate proper levels of inclusion of the NS2-specific exon in both singly spliced R1 and doubly spliced R2.     

TIA proteins are necessary but not sufficient for the tissue-specific splicing of the myosin phosphatase targeting subunit 1

We are using the tissue-specific splicing of myosin phosphatase targeting subunit (MYPT1) as a model to investigate smooth muscle phenotypic diversity. We previously identified a U-rich intronic enhancer flanking the 5' splice site (IE1), and a bipartite exonic enhancer/suppressor, that regulate splicing of the MYPT1 central alternative exon. Here we show that T-cell inhibitor of apoptosis (TIA-1) and T-cell inhibitor of apoptosis-related (TIAR) proteins bind to the IE1. Co-transfection of TIA expression vectors with a MYPT1 mini-gene construct increase splicing of the central alternative exon. TIA proteins do not enhance splicing when the palindromic exonic splicing enhancer (ESE) is mutated, indicating that TIAs are necessary but not sufficient for splicing. The ESE specifically binds SRp55 and SRp20 proteins, supporting a model in which both SR and TIA proteins binding to their cis-elements are required for the recruitment of the splicing complex to a weak 5' splice site. Inactivation of TIA proteins in the DT40 cell line (TIA-1(-/-)TIAR(+/-)) reduced the splicing of the central alternative exon of the endogenous MYPT1 as well as stably transfected MYPT1 minigene constructs. Splicing of the MYPT1 3' alternative exon and the MLC(17) alternative exon were unaffected, suggesting that TIA proteins regulate a subset of smooth muscle/nonmuscle alternative splicing reactions. Finally, reduced RNA binding and reduced expression of the TIA and SR proteins in phasic (gizzard) smooth muscle around hatching coincided with the switch from exon inclusion to exon skipping, suggesting that loss of TIA and SR enhancer activity may play a role in the developmental switch in MYPT1 splicing.     

The U1, U2 and U5 snRNAs crosslink to the 5' exon during yeast pre-mRNA splicing

Activation of pre-messenger RNA (pre-mRNA) splicing requires 5′ splice site recognition by U1 small nuclear RNA (snRNA), which is replaced by U5 and U6 snRNA. Here we use crosslinking to investigate snRNA interactions with the 5′ exon adjacent to the 5′ splice site, prior to the first step of splicing. U1 snRNA was found to interact with four different 5′ exon positions using one specific sequence adjacent to U1 snRNA helix 1. This novel interaction of U1 we propose occurs before U1-5′ splice site base pairing. In contrast, U5 snRNA interactions with the 5′ exon of the pre-mRNA progressively shift towards the 5′ end of U5 loop 1 as the crosslinking group is placed further from the 5′ splice site, with only interactions closest to the 5′ splice site persisting to the 5′ exon intermediate and the second step of splicing. A novel yeast U2 snRNA interaction with the 5′ exon was also identified, which is ATP dependent and requires U2-branchpoint interaction. This study provides insight into the nature and timing of snRNA interactions required for 5′ splice site recognition prior to the first step of pre-mRNA splicing.     

A 5' splice site-proximal enhancer binds SF1 and activates exon bridging of a microexon

Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3' and 5' splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5' splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3' splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon.